Investigation of antibacterial and anticancer activities of copper, aluminum and nickel doped zinc sulfide nanoparticles

First time compared the different metals doped ZnS nanoparticles for antibacterial and liver cancer cell line. In this study, copper, aluminum and nickel doped ZnS NPs were synthesized via co-precipitation method. The XRD analysis was confirmed the presence of cubic crystal structure and crystallite size decreased from 6 to 3 nm with doping elements. While as SEM micro-grains were revealed slightly irregular and agglomerated morphology with the presence of dopant elements. The presence of different dopant elements such as Cu, Al and Ni in ZnS NPs was identified via EDX analysis. The FTIR results demonstrate various vibrational stretching and bending modes attached to the surface of ZnS nanomaterials. After that the well diffusion method was used to conduct in-vitro bioassays for evaluation of antibacterial and anticancer activities against E.coli and B.cereus, as well as HepG2 liver cancer cell line. Our findings unveil exceptional results with maximum inhibition zone of approximately 9 to 23 mm observed against E.coli and 12 to 27 mm against B.cereus, respectively. In addition, the significant reduction in cell viability was achieved against the HepG2 liver cancer cell line. These favorable results highlight the potential of Ni doped ZnS NPs for various biomedical applications. In future, the doped ZnS nanomaterials will be suitable for hyperthermia therapy and wound healing process.

In addition, graphene base nanocomposite and functionalized graphene oxide nanocomposite (NCs) also provided the significant antimicrobial and anticancer results against different strains of bacteria and fungi and multiple type of cell line.Maruthupandy et al. 40 reported that the photocatalytic degradation of dye was improved with the help of graphene ZnO NCs.The ferrite base nanocomposites play the central role for the treatment of cancer via hyperthermia therapy and drugs delivery.While as considerable research efforts were devoted to the synthesis of nanocomposites due to their favorable biocompatibility, non-toxicity and smooth flow inside blood 21 .Likewise, silver NPs derived from the extract of Origanum Vulgare plant leaves, have exhibited notable efficacy in anticancer treatments against human liver cancer cell line (HepG2) and antibacterial activity against various bacterial strains, including drug-resistant ones 22 .After that the silver doped zinc oxide NPs coated with polyethylene glycol, have demonstrated anticancer effects on UVB-induced skin cancer cells.It also exhibited antibacterial activity against opportunistic skin pathogens 23 .The ZnS NPs were synthesized and found to inhibit the growth of MCF-7 cancer cells, while exhibiting lower toxicity compared to Stevia extract alone 24 .Additionally, ZnS NPs synthesized using a co-precipitation method with specific additives demonstrated potent antibacterial properties 25 .Moreover, ZnS NPs synthesized via sonochemical method exhibited inhibitory effects on cell migration and invasion in breast cancer cells and promoted wound healing in MCF-7 cell 26 .
In current study was focused on the synthesis of pure and doped ZnS NPs by using co-precipitation method for the evaluation of anticancer and antibacterial applications.The synthesized NPs were subjected to comprehensive characterization employing techniques including XRD, SEM, EDX, FTIR and the well diffusion method for antibacterial and anticancer activities assessment.The results revealed that Cu doped ZnS NPs exhibit enhanced antibacterial and anticancer properties displaying a significant level of effectiveness.In addition Table 1 shows the comparison of oxide and sulfide base nanomaterials for different applications.

Experiment Precursors
The high grade precursors such as zinc acetate (98%), sodium sulfate (99%), sodium hydroxide (97%), nickel acetate tetrahydrate (99.995%), aluminum acetate dibasic and copper acetate (98%) were purchase from Sigma Aldrich used for the preparation of doped zinc sulfide nanoparticles.The deionized water was purchase from local market.These nanoparticles were employed for their anticancer and antibacterial properties.

Synthesis of ZnS NPs
The ZnS NPs were synthesized with the help of sodium sulfide (Na 2 S, 0.03 M) with zinc acetate (Zn (CH 3 CO 2 ) 2 , 0.02 M) under continuous stirring on a magnetic stirrer at temperature 90 °C for 30 min.The sodium hydroxide was used to adjust the pH upto 13 (NaOH, 0.07 M).The resulting NPs were subsequently separated from the

Characterization of doped ZnS NPs
Multiple characterization techniques were used to analyze the doped ZnS NPs.The XRD analysis was utilized with Cu-Kα radiation to investigate the structural information (D8 Advance and X'Pert3 MRD XL instruments).Nova Nano (450) SEM was used to observe the surface morphology of micro grains was appeared in SEM micrograph images.The presence of elemental composition in doped ZnS NPs was examined via EDX analysis.While as FTIR Bruker spectrometer was used to identify the different functional groups attached with spectrum of undoped and doped ZnS NPs.The Fig. 1 represents the systematic diagram of synthesis methodology of undoped and doped ZnS NPs.

Antibacterial assay
The previous reported bacterial culture process was followed 42 .The antibacterial activity was tested against E.coli (ATCC 25922) gram negative and B.cereus (ATCC 14579) gram-positive by using pure; Ni, Al and Cu doped ZnS NPs.The antibacterial activity was examined by 40 mg/mL of pure; Co, Al and Ni doped ZnS NPs.The inhibition zone was observed after 24 h at 37 °C.

MTT assay of HepG2 cell line
The liver cancer cell line was cultured via 96 well plates and tissue culture flask (25 cm) was used, after that the contribute Hank salts 10% fetal bovine serum in DMEM.Furthermore, after some nonessential amino acids and glutamine 2 mM/L are incubated 24 h at 37 °C.The various concentrations (0, 10, 20, 30 and 40 mg/mL) of pure, Cu, Ni and Al doped ZnS NPs solution incorporated in cultured cells.The same MTT assay process followed as published earlier 43 .

Structural analysis
Figure 2 presents the XRD spectrum of undoped and doped ZnS NPs with diverse doping elements (Cu, Al and Ni).The XRD spectrum represents distinct diffraction peaks was attributed at various miller indices notably (111), ( 220), ( 311) and ( 331) corresponding different values of 2θ such as 28.5°, 47.8°, 56.4° and 76.9°.While as these indices represents cubic crystal structure was appeared at most prominent peak of undoped and doped ZnS NPs.Furthermore, a comparative analysis between ZnS NPs and Cu, Al and Ni doped ZnS-NPs.The observed diffraction peaks closely match the anticipated diffraction pattern for cubic ZnS ICDD powder diffraction file No. 80-0020 44,45 .There was slightly intensity decreased with Cu, Al and Ni doping agents, but the Ni incorporation into the ZnS lattice becomes evident through a discernible shift in the diffraction peak associated with the (111) plane.The corresponding 2θ value exhibits an increase from 28.86 to 29.12, owing to the smaller ionic radius of Ni (0.83 Å) compared to Zn (0.88 Å).The absence of discernible diffraction peaks corresponding to Ni, NiS, or other impurity phases validates the successful substitution of Ni as the dopant within the ZnS lattice 46 .
In addition the sharpness of the peaks decrease with the metal doping agent like Ni, it means that with the Ni doping the crystallinity of ZnS NPs decreased upto significant values.In order to examine the imperfections occurring during crystal growth, several parameters including crystallite size and peak width were computed.These calculations were undertaken to gain insights into the quality of the crystal and to evaluate the presence of any defects or distortions within its structure 47 .The Scherrer's Eq. ( 1) was preferred to examine the crystallite size of undoped, Cu, Al and Ni doped ZnS NPs.It was also investigated that the crystallite size of ZnS NPs were decreased with doping agents are expressed in (Table 2). (

Surface analysis
The surface morphology of synthesized ZnS NPs and various elements (Cu, Al and Ni) doped ZnS NPs were investigated with SEM analysis.The Fig. 3 exhibits the surface morphology of pure ZnS, Cu, Al and Ni doped ZnS NPs.The entire micrographs demonstrate that ZnS possess irregular and non-uniform grains with slight agglomeration 48 .It was also observed that the agglomeration increased with Cu doping elements in ZnS NPs.
Additionally, increase the agglomeration rate which provide evidence of the effective dispersion of Al, Ni and Cu on the surface of ZnS NPs.All the micrograph was collected 1 µm magnification level and these graph shows that flake like surface morphology was observed after doping Al, Ni and Cu in ZnS NPs.Dawngliana et al. 49 provided the information about the micrographs of ZnS NPs and also reported that nearly spherical particles was observed by calcination temperature upto 900 °C and doping of Sm +3 ion and similar morphology was observed in case of nanocomposites 49 .Figure 4 represents the identification of different elements in SEM micrographs.All the collected micrographs from (A to E) represent the different colors of Zn and sulfur and other metals doped ZnS base nanocomposite.While as all colored images were collected same magnification level 20 µm.

EDX analysis
The elemental composition and purity/clarity related information of each sample was collected by EDX analysis.
Figure 5 shows the elemental composition and weight percentage of each element in undoped and doped ZnS

FTIR analysis
Figure 6 represents the variation of rotational and vibrational modes attached on the spectrum of undoped, copper, aluminum and nickel doped ZnS NPs was identified with the help of FTIR analysis.The strong band was  appeared from 508 to 620 cm −1 indicates the existence of ZnS NPs.While as the peak was obtained at 1384 cm −1 shows nitrate group 52 .The presence of bending OH (1531 cm −1 ) and at 3350 cm -1 shows stretching OH mode.
The spectrum shows the band at 1620 cm −1 which represents CO 2 functional groups.The few other bands were observed at 1116 to 976 cm −1 which also represents Zn-S mode attached on spectrum.The same bands were reported Selvaraj and his group members in 2022 53 .In addition, Devi B et al. 35 reported similar study pure and Cu doped ZnS NPs which used starch as coating agent and all the functional groups similar to compare-able with current study.Alwany et al. 53 provided the information about lead doped ZnS NPs and the FTIR analysis indicated that the wavenumber shift toward shorter wavelength with doping agents 54 .Overall analysis shows that the current wavenumber similar to all previous reported literature of undoped, Cu and Ni doped ZnS NPs and non-availability of Al doped ZnS NPs.The Al doped ZnS NPs was first time reported in current study.Wei et al. 56 reported that the various metals doped ZnS NPs against HepG2 cell lines and previous results of ZnS sample was compared against HepG2 cell line and current study.Further, current analysis also shows that the Cu doped ZnS NCs also provided significant results against liver cancer cell line as compared to previous literature 56 .Figure 8 represents the MTT assay of Cu and Ni doped ZnS NPs.The Cu and Ni doped ZnS NPs provided most significant results as compared to pure and Al doped ZnS NPs.

Antibacterial activity
The Fig. 9 shows antibacterial activity of the synthesized ZnS NPs and (Al, Ni and Cu) doped was assessed against both E.coli and B.cereus pathogen bacteria using the well diffusion method.The study involved using the same concentration of 20 mg/mL for both ZnS NPs and those doped with Al, Ni, and Cu against the gram-negative/ positive bacterial strains.Pure ZnS NPs exhibited a minor effect on the size of the inhibition zone 9 mm for E.coli and 20 mm for B.cereus approximately.However, Fig. 9 demonstrated that the size of inhibition zone increased upto significant level by using Cu and Al doped ZnS-NPs.Notably, the maximum inhibition zone of 23 mm against E.coli and 27 mm against B.cereus was recorded for Ni doped ZnS NPs, indicating their potent antibacterial activity against both bacterial strains.Dhupar et al. 57 provided the information about indium doped ZnS NPs was used against antibacterial activity by using disc diffusion method.These analysis shows that the inhibition zone increased by increasing indium concentration in ZnS NPs 2,57 .But the current analysis shows that inhibition zone increase by varying doping concentration in ZnS NPs and every element enhance the antibacterial activity upto significant value.Figure 10 represents the inhibition zone of E.coli and B.cereus was investigated by using pure and doped ZnS NPs.Muthuchamy et al. 58 reported that the copper and ZnO NPs enhance the antimicrobial activity 58 .While as Rajivgandhi et al. 59 also provided the information CuO NPs provided less inhibition zone against P. aeruginosa and K. pneumoniae.

Conclusion
The co-precipitation method was used for the synthesis of pure, Al, Cu and Ni doped ZnS NPs.The cubic crystal structure was appeared in pure and metals doped ZnS NPs was investigated via XRD analysis.The irregular and non-uniform grains with slight agglomeration doped ZnS NPs was observed with SEM micrograph and the presence of Al, Cu and Ni in ZnS NPs was identify by EDX analysis.After that the different bending and vibrational modes attached on the surface of ZnS NPs spectrum and also identify the presence of doping agents in ZnS spectrum.After material study it was observed that the undoped and doped ZnS nanomaterial was suitable for antibacterial and anticancer activity.It was examined that with the help of well diffusion method the Cu doped ZnS NPs were provided the significant results against E.coli and B.cereus pathogen bacteria and also calculate the cell viability against liver cancer cell lines.In future the doped ZnS NPs will be used for drugs delivery and remote control process for treatment of cancer.

Figure 2 .
Figure 2. XRD spectrum of Cu, Al and Ni doped ZnS NPs.

Figure 4 .
Figure 4. SEM images for the identification of nanocomposite.

Figure 7 Figure 6 .
Figure7shows that anticancer activity of ZnS nanoparticles both undoped and doped with Cu, Al and Ni elements was evaluated against the HepG2 liver cancer cell line.Remarkably, the results demonstrated that Cu doped ZnS nanoparticles exhibited a substantial decrease in cell viability, approximately at 37 from 100%.Equally but impressive was the enhanced efficiency of Al-doped ZnS nanoparticles, showing a significant reduction in cell viability of approximately 40%.While the cell viability reduction was still considerable for pure ZnS nanoparticles (around 45%) and Ni-doped ZnS nanoparticles (approximately 40%), the remarkable performance of Cu doped ZnS NPs variants highlights their potential as promising candidates for combating liver cancer cell line55 .The bar chat which represents the value of dose (0, 10, 20, 30 and 40 mg/mL) of nanoparticles solution of undoped

Figure 7 .
Figure 7. Cell viability of doped ZnS NPs against HepG2 cell line.

Figure 8 .
Figure 8. MTT assay of Cu and Ni doped ZnS NPs.

Table 1 .
Shows the various parameters of metal oxide and sulfide base nanomaterials.
Statistical analysisData was analyzed by using two-way ANOVA and mean values of different treatments were compared by using least significant difference (LSD) at 95% confidence (P ≤ 0.05) index with the help of Co-Stat software.

Table 2 .
FWHM and crystallite size of doped ZnS NPs.